Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood, and despite much research, the genetic etiology of this complex, deadly, and widely variable disease remains unknown. This lack of knowledge is a critical obstacle to advances in predicting individual outcomes and designing more targeted, less toxic therapeutic agents. There is mounting evidence suggesting that a large degree of the variability observed in complex disease is due to inter-individual rare genetic variants. Identifying rare causal genetic variants requires deep resequencing of multiple genetic loci from a population of affected individuals, and is most informative when compared to similar sequencing from an unaffected cohort. Traditional dideoxy sequencing methods are too cost and time prohibitive for such large scale analyses. However, by applying a targeted, population-based pooled DNA sequencing method that we have designed to leverage the massively parallel, high-throughput capacity of next-generation sequencing, we propose to deeply resequence 56 genes implicated in pediatric high-risk leukemogenesis from the germline DNA of 96 unaffected children and 96 matched non-tumor and tumor DNA from pediatric high-risk ALL patients. While a specific causal rare variant is individually rare, a disease-associated genetic locus, when interrogated on a population-based scale, might demonstrate a variety of different rare variants all resulting in a similar phenotype. We expect to identify such loci in our pediatric ALL cohort. We will confirm these results in a validation cohort of 250 pediatric high-risk ALL patients followed by individual genotyping of validated loci to identify potential inter-individual gene-gene interactions. We expect these results to identify a variety of genes and biochemical pathways involved in pediatric leukemogenesis which would then form the basis for long-term cell based functional study, with the ultimate goal of providing additional data that could be used for prospective individual patient genotyping for risk assessment, individualized therapy, and design of new targeted therapeutic agents. PUBLIC HEALTH RELEVANCE: There is increasing evidence that unique individual combinations of rare genetic changes may account for a significant degree of variability in the susceptibility and treatment response of complex disease, particularly pediatric leukemia. We have designed a new high-throughput, targeted, cost-effective method for DNA sequencing. By applying this method and sequencing dozens of genes important in pediatric leukemia, I expect to rapidly identify a variety of new genetic changes and biochemical pathways that, when altered, may predispose a child to developing leukemia at such a young age.